ICAM-1-binding Plasmodium falciparum erythrocyte membrane protein 1 variants elicits opsonic-phagocytosis IgG responses in Beninese children

Members of the highly polymorphic Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on the surface of infected erythrocytes (IEs) are important virulence factors, which mediate vascular adhesion of IEs via endothelial host receptors and are targets of naturally acquired immunity. The PfEMP1 family can be divided into clinically relevant subgroups, of which some bind intercellular adhesion molecule 1 (ICAM-1). While the acquisition of IgG specific for ICAM-1-binding DBLβ domains is known to differ between PfEMP1 groups, its ability to induce antibody-dependent cellular phagocytosis (ADCP) is unclear. We therefore measured plasma levels of DBLβ-specific IgG, the ability of such IgG to inhibit PfEMP1-binding to ICAM-1, and its ability to opsonize IEs for ADCP, using plasma from Beninese children with severe (SM) or uncomplicated malaria (UM). IgG specific for DBLβ from group A and B ICAM-1-binding PfEMP1 were dominated by IgG1 and IgG3, and were similar in SM and UM. However, levels of plasma IgG inhibiting ICAM-1-binding of group A DBLβ of PFD1235w was significantly higher in children with UM than SM, and acute UM plasma induced a higher ADCP response than acute SM plasma.


Scientific Reports
| (2022) 12:12994 | https://doi.org/10.1038/s41598-022-16305-0 www.nature.com/scientificreports/ subgroup of group A and B/A PfEMP1 proteins can bind both to the intercellular adhesion molecule 1 (ICAM-1) and endothelial protein receptor C (EPCR), and this dual-binding phenotype has been linked to CM 14,15 but not all studies 16 . Infected erythrocytes co-localize with ICAM-1 expression in the brain blood vessels suggesting that ICAM-1 mediates IE sequestration in CM 17 . Dual-receptor binding IEs have been shown in vitro to cause clustering of ICAM-1, to be taken up by brain endothelial cells in an ICAM-1-dependent manner, that results in the breakdown of the blood-brain barrier and swelling of endothelial cells, and antibodies directed to the PfEMP1 on their surface prevent sequestration of IEs 18 . Sequestration of large numbers of IEs in the microvasculature of specific organs is central to the pathogenesis of severe P. falciparum malaria 19 . CIDRα domains associated with CM 20,21 are the focus of some research groups, while we focus on DBLβ domains. In this study, we hypothesized that children with uncomplicated malaria (UM) have higher levels of functional anti-PfEMP1 (anti-DBLβ domain) antibodies than severe malaria (SM) cases and because of this, they would have antibodies against PfEMP1 variants associated with severe disease. To test this, we measured plasma levels of anti-PfEMP1 (DBLβ)-IgG in a cohort of Beninese children with SM or UM and used ICAM-1-binding inhibition and ADCP as measures of antibody effector function.

IgG specific for DBLβ domains in ICAM-1-binding PfEMP1 variants. To investigate the anti-
PfEMP1-IgG antibody reactivity against ICAM-1-binding DBLβ domains, 137 Beninese children (median age 36 months; IQR 22 to 48 months) were recruited at three different hospital centers and divided into three clinical categories: cerebral malaria (CM), non-cerebral severe malaria (nCSM), and uncomplicated malaria (UM) ( Table 1). The mortality rate of CM cases was 26.5% versus 2% among children with nCSM or UM. The mean hemoglobin level of UM was significantly higher than that of children with CM or nCSM (P < 0.0001, Table 1). We assessed IgG levels against seven group A and four group B ICAM-1-binding DBLβ domains, and against three non-ICAM-1-binding group A DBLβ domains (Fig. 1). The anti-DBLβ-IgG levels among the children were significantly higher at hospitalization (day 0) than at convalescence (day 30) for four group A ICAM-1-binding DBLβ domains (PF11_0521, Dd2VAR32, KJ866957, KM364031), two group B ICAM-1-binding domains (IT4VAR13, PFL0020w) and two group A non-ICAM-1-binding PfEMP1 domains (Dd2VAR25, Dd2VAR52) ( Fig. 1). We did not find any significant differences in reactivity between SM (CM and nCSM) and UM (Fig. 2).

Subclass IgG antibody responses.
To determine whether the antibody subclass response against different ICAM-1-binding DBLβ domains differed, we measured both cytophilic (IgG1, IgG3) and non-cytophilic (IgG2, IgG4) IgG in plasma samples from 84 (day 0) and 45 (day 30) of the children. Cytophilic IgG completely dominated the anti-DBLβ (PFD1235w, PF11_0521, HB3VAR03, HB3VAR21, PFL0020w, IT4VAR13) antibody response in all children (Fig. 3), as IgG2 and IgG4 levels were low to undetectable (data not shown). The dominance of IgG1 and IgG3 suggests that antibody-mediated protection against SM relies not only on inhibition of IE sequestration (neutralization), but also involves ADCP and possibly activation of the classical complement cascade. Levels of HB3VAR03-specific IgG3 were significantly higher in children with SM (day 0, P = 0.04) compared to children with UM (Fig. 3c). A similar trend was seen for PFD1235w-specific IgG3, but this did not reach statistical significance (P = 0.09) (Fig. 3a). The IgG1 and IgG3 antibody response against PF11_0521 (group A), and group B DBLβ domains (HB3VAR21, PFL0020w, IT4VAR13) did not differ between the two clinical categories (Fig. 3b,d-f).  . 4a1-a4). The ability to inhibit binding of the PfEMP1 domains to ICAM-1 did not correlate significantly with the IgG antibody level against the specific DBLβ domain (Fig. 4b1-b4). From this analysis, we proceeded to stratify our study participants into the three clinical categories CM, nCSM, and UM to assess the potential inhibitory role of their plasma (Fig. 4c). Although the median anti-PFD1235w IgG level of the different disease categories did not differ significantly (Fig. 3a), the median percentage plasma IgG inhibition of PFD1235w DBLβ binding to ICAM1 (Fig. 4c1) was significantly higher for UM than nCSM (P = 0.001) Antibody-dependent cellular phagocytosis. ADCP of merozoites has been suggested to contribute to protective immunity in humans, and the opsonic phagocytosis assay has been shown as a valuable technique to assess anti-malarial immunity 22 . We optimized a bead-based ADCP assay from the protocol described by Lloyd et al. 23 using undifferentiated CD64 + (FcγRI), CD32 + (FcγRII) and CD16 -(FcγRIII) THP-1 cells (Fig. S1). Neutravidin and fluorescently labelled beads coupled with biotinylated DBLβ protein (PFD1235w, HB3VAR03, HB3VAR21, IT4VAR13) domains were pre-incubated with plasma from our Beninese donors (positive controls, i.e., a pool of highly reactive samples from Beninese individuals) or from negative control donors without exposure to P. falciparum parasites. THP-1 cells were added, and bead uptake by the cells was estimated by flow cytometry (Fig S2). The presence of malaria-specific IgG had no or little effect on the uptake of uncoupled BSAbeads nor did the presence of malaria naïve IgG affect the uptake of coupled beads by THP-1 cells (Fig. S2b). This demonstrates that the phagocytosis of beads by the THP-1 cells depended on opsonization by antigenspecific IgG. Having established the optimal dilution of plasma to be 1:200 (Fig. S2g, h), we measured the functional activity of antigen-specific plasma IgG antibodies. Except for HBVAR03 DBLβ (P = 0.68), the convalescent (day 30) Beninese plasma samples promoted a stronger phagocytosis of DBLβ-coated beads (PFD1235w and IT4VAR13 DBLβ, P < 0.0001) than samples obtained on the day of hospitalization (Fig. 5a1-a4). This was also seen for matched samples ( Fig. 5b1-b4).

Adhesion-inhibitory plasma
The anti-DBLβ IgG levels against PFD1235w and HB3VAR03 (group A), and against IT4VAR13 (group B), were positively correlated with the ADCP activity against the DBLβ domains (PFD1235w, r = 0.48, P < 0.001; HB3VAR03, r = 0.50, P < 0.001; IT4VAR13, r = 0.39, P < 0.001), suggesting that this type of PfEMP1-variants are www.nature.com/scientificreports/ major targets of IgG promoting ADCP ( Fig. 5c1-c4). To further evaluate the significance of PfEMP1 as a target of acquired functional antibodies, and to examine whether responses to specific PfEMP1 domains might be important in protection from severe disease, we stratified the data based on the clinical category of the children. The level of IgG to each of the four DBLβ domains (PFD1235w, HB3VAR03, HB3VAR21, IT4VAR13) were similar in the UM and SM (Fig. 3), however in the functional assay, most samples from UM children showed a marked increase in phagocytosis activity against IT4VAR13 compared to those from SM children at day 0 ( Fig. 5d4), but this did not differ for PFD1235w, HB3VAR03, and HB3VAR21 ( Fig. 5d1-d3).

Discussion
Plasmodium falciparum causes the most severe form of malaria and expresses PfEMP1 proteins on the surface of IEs. These proteins are expressed in a mutually exclusive manner and adhere to a range of vascular receptors that facilitate IE evasion of splenic clearance 24,25 . Severe malaria has been linked to IE adhesion via specific host receptors and is mediated by structurally related PfEMP1 (reviewed in 11,26 ). In malaria endemic areas with stable transmission of P. falciparum, protective immunity is acquired during childhood, first to severe complications and later to clinical disease 27,28 . This is believed to be a result of an ordered acquisition of antibodies, with antibodies to a relatively conserved set of PfEMP1 proteins associated with severe disease acquired prior to antibodies to a larger and more diverse set of PfEMP1 proteins associated with uncomplicated malaria and asymptomatic parasitemia 27,29 .
In Beninese children, we found that IgG reactivity against ICAM-1-binding DBLβ did not differ among children of similar age with severe or uncomplicated malaria (Fig. 2), and that IgG levels were reduced 30 days following hospitalization (Fig. 1). In agreement with this, our recent study found that Ghanaian children show a transient increase in IgG reactivity to ICAM-1-binding DBLβ domains two weeks after acute malaria followed  Fig. 1 were grouped according to clinical disease category, i.e., severe malaria (SM including CM and nCSM; n = 28), and uncomplicated malaria (UM, n = 27) at the day 0 (hospitalization) and day 30 (convalescent) samples. The reactivity was measured against the same DBLβ domains as in Fig. 1 www.nature.com/scientificreports/ by decreased levels six weeks later. As in the present study, our previous work 30 and that of others 16,31 did not observe differences in IgG reactivities to DBLβ domains between children of similar age with severe or uncomplicated malaria.
The clinical significance of PfEMP1-specific antibodies is thought to involve their ability to interfere with sequestration of IEs in various tissues including DBLβ-specific-IgG that inhibit IE adhesion to ICAM-1 9,14,32,33 . Here, we investigated the activity of naturally acquired functional antibodies from children who were exposed to P. falciparum infection, i.e., we looked at adhesion-inhibitory antibodies and antibodies mediating ADCP. Our findings show that antibodies from participants included in the study were able to not only react with the domains, but that the IgG antibodies have functional activity as they elicited inhibitory effects ranging from 16 to 78% upon the ICAM-1-binding of DBLβ domains encoded by dual-receptor binding PfEMP1 (Fig. 4), and www.nature.com/scientificreports/ promoted opsonic phagocytosis (Fig. 5). These findings support previous work, which shows that the PfEMP1 family is an important target of protective antibodies against malaria 5,10,34-37 . When stratifying our study participants into distinct clinical categories to assess the potential inhibitory role of their antibodies, plasma from children with UM showed higher inhibition of PFD1235w DBLβ binding to ICAM-1 compared to plasma from SM children, including CM cases (Fig. 4). Thus, anti-PFD1235w IgG antibodies can inhibit PfEMP1 binding and might prevent PFD1235w-expressing IEs (or close variants thereof) from binding to ICAM-1 on endothelial cells, thus providing protection against developing SM in these children. if that hypothesis can be confirmed in future studies, it would indicate that inclusion of peptides based on this domain in malaria vaccine cocktails would be warranted. Cytophilic antibodies, IgG1 and IgG3 are known for their high affinity for most of the Fc receptors on diverse immune cells and their function in opsonization for effector cell function [38][39][40][41] . As in other studies 42,43 , the IgG antibody response was dominated by IgG1 and IgG3 (Fig. 3), which facilitate protection against malaria through cell-mediated mechanisms, such as ADCP and antibody-dependent cellular inhibition. In contrast, IgG2 and IgG4 have been classically considered as non-protective antibodies against malaria because they poorly engage Fc receptors 41 , and levels of PfEMP1-specific IgG of these sub-classes IgG2 and IgG4 isotype levels were low in our study participants (data not shown). Besides neutralization of pathogens by antibodies, phagocytosis is considered as one of the most important anti-pathogen activities 44 . ADCP involves receptor-mediated interactions between immune effector cells and the Fc domain of cytophilic IgG bound to pathogen 45 . Here, we measured opsonic phagocytosis activity using undifferentiated THP-1 monocytes and found that children with UM and SM had similar levels of IgG to the different DBLβ domains, but plasma from UM children (day 0) showed significantly higher percentage phagocytosis activity to group B IT4VAR13 DBLβ (Fig. 5d4). This could indicate that a higher opsonic phagocytosis activity of plasma against DBLβ domain is associated with protection from developing SM, a finding that will be of interest to investigate further in future studies.

Methods
Plasma and parasite samples. The study protocol was reviewed and approved by the Comité National d'Ethique pour la Recherche en Santé (CNERS), No. 87/MS/DC/SGM/DRFMT/CNERS/SA Cotonou, République du Benin. All methods used in this study were performed in accordance with relevant guidelines and regulations. Plasma samples (n = 137) were collected at three hospital centers in the cities of Cotonou, Southern Benin during the malaria transmission season of June-September 2019. After informed consent had been obtained from a parent or a legal guardian, children less than 5 years of age were subjected to clinical investigation and screened by malaria rapid diagnostic test (Malaria Pf/Pan, DiaQuick). Venous blood samples were collected from the enrolled children and parasitemia quantified by microscopy. Blood samples were collected from each child on the day of hospitalization (disease presentation) and 30 days following discharge (convalescent sample). Clinical manifestations were classified according to the definitions by the World Health Organization 46 . Patients were categorized as having cerebral malaria (CM; n = 34) if they had a positive blood smear of P. falciparum and unarousable coma (Blantyre coma score [BCS] ≤ 2) with exclusion of other causes of coma. Patients were categorized as having non-cerebral severe malaria (nCSM; n = 51) if they presented with hyperparasitemia (> 50,000), and/or severe anemia (hemoglobin < 5 g/dL), and no coma. Patients with uncomplicated malaria (UM; n = 52) had fewer parasites than 50,000 parasites per µL accompanied by fever, headache, or myalgia without signs of severity and evidence of vital organ dysfunction.
A pool of plasma from P. falciparum-exposed Liberian adults 47 , or hyperimmune Beninese children, and 30 non-exposed Danish individuals were used in ELISA as positive and negative controls, respectively.
The P. falciparum parasite clones PFD1235w, HB3VAR03, IT4VAR13 and HB3VAR21 (KOB63129) were maintained in long-term in vitro culture and selected for IE surface expression by repeated antibody selection as described [48][49][50] . The identity of isolates was routinely verified by genotyping as described 51 (Table S1) were purified by immobilized metal ion affinity chromatography using HisTrap HP 1-ml columns (GE Healthcare) as described previously 27,30,32 . Recombinant Fc-tagged ICAM-1 was expressed in HEK293 cells and purified as described previously 52 .   www.nature.com/scientificreports/ Biosciences), and THP-1 cells were gated by size, and granularity on FSC and SSC plots and fluorescence in the FITC-channel. A minimum of 5,000 THP-1 cells were recorded for each sample. Data were analyzed using the FlowJo (ver 10.6.0) software. Percentage phagocytosis was the proportion of THP-1 cells that were positive for fluorescence in the FL-1 channel. THP-1 cells in two major fluorescence peaks were visualized by setting the FL-1 channel against the FL-4 channel.

Statistical analysis.
Wilcoxon rank sum test was used to compare antibody levels in matched and paired data groups between the time of hospitalization and at convalescence. We used Kruskal-Walli's test with Dunn´s multiple comparison test of functional data for children in different clinical categories i.e., CM, nCSM, and UM. Differences were considered significant when P < 0.05. GraphPad Prism 9.0 was used for the analysis.

Data availability
The datasets used and analyzed during the current study are available from the corresponding author on request.